Effect of sorghum flour on the glutathione bread First, we investigated the effect of sorghum flour on the glutathione-rice batter in terms of the swelling of the bread.
The total amount of the flour was 160 g with various rice/
sorghum flour ratios. The amount of GSSG and distilled water was 0.5 g and 140 g, respectively. Figure 1 shows the specific volume and a cross-section of the breads. The specific volume was the highest (3.79 ± 0.11 cm3/g) and the crumbs appeared finest when the rice/sorghum ratio was around 130/30. Raising the portion of sorghum flour further did not improve the specific volume or crumb appearance (data not shown).
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Fig. 2 Effect of oxidized glutathione on the sorghum/rice bread
A, The specific volume of breads plotted against the amount of oxidized glutathione (GSSG) added. Each data point represents the mean
± SD of three independent experiments. B, Cross-sections of some of the breads. The amounts of GSSG were 0, 2, 5, and 10 g for a, b, c, and d, respectively. C, Low-vacuum scanning microscopic analysis of the control (a) and GSSG-added breads (b). The amounts of sorghum flour, rice flour, distilled water, dried yeast, and sugar were 30, 130, 140, 2.5, and 15 g, respectively. The standard fermentation and baking conditions were 40°C for 60 min and 180°C for 24 min, respectively.
Amount of GSSG (g) against 160g flour Specific volume (cm3 /g)
A
B a b
c d
C a b
100µm 100µm
4 3 2 1 0 3.5 2.5 1.5 0.5 4.5 5
1 2 3 4 5 6 7 8 9 10 11 12 0
Fig. 3 Effect of sorghum flour on the salt tolerance of the rice bread containing glutathione
A, Cross-sections of the relevant rice breads. a, Rice bread; b, Oxidized glutathione (GSSG)-containing rice bread; c, Sorghum/rice bread; d, GSSG-containing sorghum/rice bread. B, The specific volume of each bread. Each data point represents the mean ± SD of three independent experiments. The amount of GSSG for b and d was 1 g. The amount of rice flour was 160 g for a and b. The amounts of sorghum flour and rice flour were 30 and 130 g, respectively, for c and d. The amounts of salt, distilled water, dried yeast, and sugar were 2.4, 140, 2.5, and 7.5 g, respectively, for a to d. The standard fermentation and baking conditions were 40°C for 60 min and 180°C for 24 min, respectively.
4 3 2 1 0 3.5 2.5 1.5 0.5
a b
4.5c d
a b c d
B A
Specific volume (cm3/g)
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Effect of oxidized glutathione on the sorghum/rice bread
Next, we investigated the effects of GSSG on the sorghum/rice batter in terms of the specific volume and crumb coarseness/fineness of the bread. The amount of rice/
sorghum flour was 130 g/30 g, as this ratio provided the highest specific volume in the previous experiment (Fig.
1). Figure 2A shows the specific volume of breads plotted against the concentration of glutathione. Interestingly, the specific volume was relatively high (3.40 ± 0.04 cm3/ g) even in the absence of GSSG. The maximum specific volume, around 4.1 cm3/g, was obtained with addition of 1 to 2 g of GSSG against 160 g of the flour mixture.
When the amount of GSSG was 3 g or higher, the specific volume decreased. Figure 2B shows a cross-section of the rice/sorghum bread with and without GSSG. The crumbs appeared coarse in the absence of GSSG but became finer in its presence. The low-vacuum scanning microscopic analysis (Fig. 2C) of the control (a) and GSSG-added (b) breads suggested that the bubble wall was thicker in the absence of GSSG and thinner in its presence. Thus, it was speculated that the combined use of sorghum/rice flour strengthens the framework of the bubbles by thickening
their walls.
The enhancing effect of the use of mixed flours on the volume of gluten-free bread has already been reported (Sanchez and others 2002; Sciarini and others 2010).
The mixture of sorghum/rice flour should have a similar effect. On the other hand, glutathione seems to enhance the fineness of the crumb structure. Although the swelling mechanism of the glutathione bread has not yet been elucidated, we hypothesize that glutathione affects the structure of the batter proteins by reducing the protein disulfide bonds (Yano and others 2013). Hamaker and Griffin (1993) have proposed that an endosperm matrix protein, or possibly a specific starch-granule-associated protein, exerts influence on the gelatinization behavior of rice starch granules. In short, the barrier theory postulates that disulfide-bound protein polymers in or surrounding the native starch granule affect the viscoelastic properties of the cooked grain and flour. When the protein disulfide bonds are disrupted, rice starch granules swell to a larger size, thereby increasing the paste viscosity. Therefore, as we discussed earlier, the addition of glutathione may cleave the disulfide bonds of the barrier protein, thereby increasing the batter viscosity as well as the gas-holding capacity in fermentation Fig. 4 Effects of a yeast extract with a high content of oxidized glutathione on the sorghum/rice bread A, Cross-sections of the control (a) and yeast extract-containing (b) sorghum/rice breads. B, Sensory test of the breads with respect to crumb appearance (left) and preference (right). The results of the sensory tests (i.e., the difference in the crumb fineness and preference of the bread containing YH-D18 compared with the control bread) are shown. The ingredients of the breads were sorghum flour (30 g), rice flour (130 g), water (140 g), sugar (15 g), dried yeast (2.5 g), and butter (2 g). Two grams of the yeast extract YH-D18 had been added to the batter in panel b only. The standard fermentation and baking conditions were 40°C for 90 min and 180°C for 24 min, respectively.
A
B
0 +1 +2 +3
Appearance Preference Much finer
Finer Slightly finer None
Much better Better Slightly better None
a b
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(Yano 2010). Meanwhile, in their report on protease-treated rice batter, Hamada and others (2013) considered that a partially denatured protein, possibly glutelin, may work as a linker to join starch granules. These aggregates of starch granules, which have a lot of space within them, have a lower specific gravity and result in slower sedimentation. Hamada and others (2013) concluded that the aggregates may have caused a change in the batter rheology, particularly its viscosity and sedimentation behavior. Our own ongoing studies, as well as those of other researchers, will reveal the relations between the barrier protein and starch granule, as well as the swelling mechanism of the gluten-free bread.
Effect of sorghum on the salt tolerance of glutathione-rice bread
One of the major challenges in developing glutathione-rice bread is that salt reduces the swelling of the batter in fermentation (Yano 2010). This is an important problem to overcome, since the taste of salt is a key factor in the preference of bread (Miller and Hoseney 2008). We therefore investigated whether the addition of sorghum flour to the glutathione-rice batter would enable the batter to rise during fermentation, even in the presence of salt.
Figure 3A shows a cross-section of the relevant rice breads that contained 2.4 g of salt in 160 g of sorghum/rice flour.
Figure 3B shows the specific volume of each type of bread.
Without sorghum flour, the specific volumes of the rice bread were 2.28 ± 0.15 cm3/g and 2.54 ± 0.12 cm3/g in the absence (a) or presence (b) of GSSG, respectively. On the other hand, addition of sorghum flour improved the swelling of the batter during fermentation, increasing the specific volume of the breads to 3.49 ± 0.09 cm3/g and 3.82
± 0.09 cm3/g in the absence (c) or presence (d) of GSSG, respectively. The crumbs appeared finer in the presence of GSSG (compare Fig. 3A c and d), even in the presence of salt. Conclusively, the addition of sorghum flour to the glutathione-rice batter was effective in making the bread swell and the crumb structure finer, even in the presence of salt.
Substitution of a yeast extract with high GSSG content for purified GSSG
Next, we investigated whether purified GSSG could be replaced with a yeast extract with a high concentration of GSSG. Although purified glutathione can be used as a food
additive in the USA and several Asian countries, it cannot be used as a food material in Europe and Japan. Glutathione is produced on an industrial scale by yeast fermentation (Li and others 2004). Although the commonly used yeast extracts contain only a negligible amount of reduced glutathione (GSH) or GSSG, specific yeast extracts with a high content of GSH or GSSG glutathione are commercially available. In many countries, it is permissible to use such yeast extracts as an ingredient in foods. In the following experiment, we used the yeast extract YH-D18, which contains approx. 18% (w/w) of GSSG.
Figure 4A shows cross-sections of sorghum/rice breads with a rice/sorghum ratio of 130/30. No GSSG had been added to the bread in panel a (control), whereas 2 g of yeast extract YH-D18 had been added to the bread in panel b. Rice batter with YH-D18 did not swell in the absence of sorghum flour (data not shown). The specific volume of the control and the YH-D18-containing bread was 3.34
± 0.08 cm3/g and 3.74 ± 0.09 cm3/g, respectively. The results were consistent with the observed effects of purified GSSG addition, which also increased the specific volume of sorghum/rice bread (Fig. 2A). It also appeared that the addition of the yeast extract made the breadcrumbs finer, as shown by the addition of purified GSSG (Fig. 2B).
Next, the breads were subjected to a comparative sensory test by a panel of 12 trained tasters employed at the Japan Food Research Laboratories (Tokyo, Japan), a foundation that offers analytical services on food products.
Figure 4B shows the difference in the crumb fineness/
coarseness between the bread containing YH-D18 and the control bread without YH-D18 as judged by the panel, with the results being scored on a 7-point scale (-3 to +3). The crumbs of the bread containing YH-D18 were recognized as being finer than those of the control bread. The difference was 1.33 ± 0.75 (p < 0.01) between “finer” and “slightly finer.” Moreover, when masticated, the bread containing YH-D18 was judged as being preferable (1.42 ± 0.76;
p < 0.01) to the control bread; that is, between “better”
and “slightly better.” The sensory tests thus conclusively demonstrated a clear effect of YH-D18 addition on the fineness of the breadcrumbs. It was speculated that this difference was attributable to the GSSG contained in the yeast extract YH-D18, since the results were consistent with those obtained using purified GSSG, as shown in Figures 2 and 3. Thus, a yeast extract with a high content of GSSG
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or GSH may be useful for modifying the disulfide structure of food proteins. As disulfide bonds play critical roles in food chemistry (Yano 2014), this finding may expand the use of yeast extract in the food industry. Meanwhile, as the yeast extract contains other components, such as proteins, amino acids, lipids, and carbohydrates, some of these components might have exerted a secondary action on the swelling during fermentation or on the crumb fineness.
Therefore, further studies will be needed to confirm a causal relationship between the GSSG in YH-D18 and the improvement of the bread quality.
Collectively, the studies performed in this and previous reports have completed the basic formulation for a glutathione-based gluten-free bread. This bread can be made using foodstuffs only; that is, rice flour, sorghum flour, yeast extract with a high content of glutathione, salt, dry yeast, sugar, and water. No specific apparatus is required other than a commercially available home bakery and electric oven. Semisynthetic chemicals such as HPMC or guar gum are not needed. Collaborative studies with bread makers to improve the quality of the bread and thus enhance its practical applicability as a food are ongoing in our laboratory. Hamaker and others (1987) reported that although cooked sorghum protein is less digestible than other cooked cereal proteins, cooking sorghum in the presence of reducing agents such as 2-mercaptoethanol, dithiothreitol, sodium bisulfite, and l-cysteine increased the protein digestibility to a level comparable with other cereals. Thus, studies on the effect of glutathione on the nutritional value of the bread should also be conducted.
Finally, it will be necessary to verify whether the bread has reduced malignancy in celiac and wheat allergy patients.